Behaviors were compared between saline and amphetamine
Behaviors were compared between saline and amphetamine pre-treatment groups at 24h and 2 weeks following the last treatment, or between pre-treatment groups that received vehicle or ASV-30 infusions, using separate two-way ANOVAs. Significant effects were analyzed further using Student–Newman–Keuls post-hoc test for multiple pair-wise comparisons. Significance levels for all statistical tests were set at ≤0.05 (SigmaStat v2.03, SPSS Inc., Point Richmond, CA, USA). When anxiety-like behavior during amphetamine withdrawal was assessed, latency to enter open arms was significantly affected by pre-treatment treatment (=8.338, =0.007), but there was no effect of withdrawal Actinonin (=0.173, =0.680), nor an interaction between withdrawal period and pre-treatment (=0.665, =0.421). A significant increase in latency was observed between treatment groups only within the 2-week withdrawal group (SNK <0.05; A). Similarly, time spent in open arms was significantly different between pre-treatment groups (=25.850, <0.001) but was not affected by withdrawal period (=0.0421, =0.839), and an interaction between pre-treatment and withdrawal period was not observed (=0.152, =0.699). Time spent in the open arms was significantly decreased in the amphetamine treatment groups compared to saline-treated rats within both the 24-h and 2-week withdrawal periods (SNK <0.05; B). There was no effect of treatment (=0.586, =0.449), withdrawal (=3.561, =0.068), nor an interaction between treatment and withdrawal (=0.362, =0.551) on the total distance traveled in the maze (C). For experiments that tested CRF receptor mediation of amphetamine-induced anxiety-like behavior, placement of infusion cannulae into the dRN did not differ between saline and amphetamine pre-treated rats (A and B). A significant effect of pre-treatment (=6.155, =0.021), dRN infusion (=5.900, =0.023), and a significant interaction between pre-treatment and dRN infusion (=4.916, =0.036) was observed in the latency to enter open arms. Amphetamine pre-treated rats infused with vehicle showed greater latency to enter open arms compared with saline pre-treated rats and amphetamine pre-treated rats infused with ASV-30 (SNK <0.05; C). For time spent in opens arms, there was an effect of dRN infusion (=6.715, =0.016) and an interaction between drug pre-treatment and dRN infusion (=9.686, =0.005). Amphetamine pre-treated rats infused with vehicle showed reduced time in open arms compared with saline pre-treated rats and amphetamine pre-treated rats infused with ASV-30 (SNK <0.05; D). There was no significant effect of pre-treatment (=0.296, =0.592), dRN infusion (=0.00752, =0.932), nor an interaction of pre-treatment and dRN infusion (=0.0108, =0.918) on total distance traveled in the maze (E). Overall, our results show that rats exhibited heightened anxiety-like behaviors both 24h and 2 weeks following the last repeated injection of amphetamine. The distance traveled in the EPM was nearly identical between saline- and amphetamine-treated groups at both time points measured, suggesting that differences observed in anxiety-like measures were not due to differences in the activity between the treatment groups. Therefore, these findings suggest that chronic amphetamine treatment increases anxiety states, which persist during protracted drug withdrawal. Infusion of a CRF receptor antagonist directly into the dRN reduced anxiety-like behaviors exhibited by amphetamine pre-treated rats at 2 weeks of withdrawal, with no effect on locomotion within the maze. These results extend those of Pringle et al. showing increased CRF receptors in the dRN up to 6 weeks following 14 days of amphetamine treatment, and of Sarnyai et al. and Basso et al. demonstrating increased anxiety-like behaviors of rats at 48h of withdrawal following 14 days of cocaine treatment, which were ameliorated by icv CRF antiserum or a CRF receptor antagonist. Combined, these findings suggest that one of the mechanisms by which chronic psychostimulant treatment and withdrawal results in elevated anxiety states may be increased CRF receptor activity in the dRN. Given that CRF receptor activation within the dRN leads to increases in 5-HT release in limbic regions such as the amygdala and nucleus accumbens , , heightened CRF receptor activity in the dRN during amphetamine withdrawal may amplify serotonergic activity within the limbic system to result in increased anxiety states.